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Volume 272, Number 19, Issue of May 9, 1997 pp. 12300-12304
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Ribosomal Binding Site of Release Factors RF1 and RF2
A NEW TRANSLATIONAL TERMINATION ASSAY IN VITRO

(Received for publication, December 4, 1996, and in revised form, January 30, 1997)

Guido Grentzmann and Paul John Kelly ^¶

From the  Eccles Institute of Human Genetics, Howard Hughes Medical Institute and ^¶ University of Utah, Salt Lake City, Utah 84112

We have established a new in vitro assay for translational termination. It consists of 70 S ribosomes bound to a synthetic RNA minimessenger via interaction with P-site binding fMet-tRNA_f^Met. If the A-site codon is a stop signal, release activity can be measured by quantifying hydrolyzed formylmethionine. Characteristics of this assay in terms of reaction time, ion concentration, release factor RF1 and RF2 concentration, and competition with A-site-decoding tRNA are discussed. The new assay shows that polypeptide chain release activity is directly dependent on the presence of a stop codon in the ribosomal A-site.

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